Herpetological Conservation and Biology 9(1):170–180. Submitted: 10 October 2012; Accepted: 22 January 2014; Published: 13 July 2014.

REPRODUCTIVE BIOLOGY OF PHYMATURUS SPECTABILIS (LIOLAEMIDAE): FEMALES SKIP REPRODUCTION IN COLD AND HARSH ENVIRONMENTS OF PATAGONIA, ARGENTINA

1,5 1 2 JORGELINA M. BORETTO , FACUNDO CABEZAS-CARTES ,FABIÁN TAPPARI , FAUSTO 3 4 2 MÉNDEZ-DE LA CRUZ , BARRY SINERVO , ALEJANDRO J. SCOLARO , AND NORA R. 1 IBARGÜENGOYTÍA

1INIBIOMA (CONICET – Universidad Nacional del Comahue). Bariloche, Río Negro, Argentina, e-mail: [email protected]; [email protected] 2Universidad Nacional de la Patagonia, Trelew, Chubut, Argentina, e-mail: [email protected] 3Laboratorio de Herpetología, Instituto de Biología, Universidad Nacional Autónoma de México, D.F. 04510, México, e-mail: [email protected] 4Department of Ecology and Evolutionary Biology, Earth and Marine Sciences Building, University of California, Santa Cruz, California 95064, USA, e-mail: [email protected]

Abstract.—Herein, we describe the reproductive biology of Phymaturus spectabilis, a lizard species that inhabits rocky outcrops in cold and arid environments of the Patagonian steppe of Argentina. We studied male and female reproductive cycles, mean annual reproductive output, allometric changes during ontogeny, and sexual dimorphism, mainly with a non-invasive methodology used for the first time in Argentina. We corroborated results from this non-invasive method with data from euthanized individuals through observation, classification, and measurement of the female reproductive tract and ovaries and through examination of the histology of testicles and epididymis in males. The presence of males with spermatozoa in the epididymis from early spring and gravid females from mid spring shows that mating, ovulation, and egg development occur at the beginning of the activity season. Parturition occurs in midsummer to two offspring. Our finding that half of the adult females were in a non-reproductive condition during the entire reproductive season, simultaneously with vitellogenic or gravid females, demonstrates that not all of the adult females in this population reproduce every year. Females exhibited an annual to biennial cycle, sometimes skipping a year of reproduction, while males exhibited an annual cycle. Phymaturus spectabilis show the common pattern of low mean annual reproductive output found in the genus Phymaturus. The low frequency of reproduction and the low mean annual reproductive output are risk factors for this species. These life-history traits, together with its microendemic character (as it is restricted to an area of 70 km2 of fragmented and rocky plateaus in the poorly protected steppe of Patagonia Argentina), mean that this species is of concern.

Key Words.—abdominal palpation; cold and harsh steppe; lizards; reproduction

INTRODUCTION under study that may represent new species (Lobo et al. 2012; Morando et al. 2013). However, the Reproductive biology studies are essential for reproductive biology of the majority of these species is understanding the life history of a species and have a unknown. direct relevance for conservation. This is especially Species of the genus Phymaturus show features true in the lizards of the Phymaturus genus conserved from the ancestral state, such as their (Liolaemidae). Although the reproductive biology of consistent preference for rocky microhabitats, a only six Phymaturus species has been studied, their mainly herbivorous diet, and a viviparous reproductive reproductive characteristics and low mean annual mode (Cei 1986, 1993; Scolaro and Ibargüengoytía reproductive output, together with characteristics of 2008). The cold and harsh environmental conditions their ecology and distribution, make them vulnerable restrict the activity of Phymaturus lizards to a limited (Abdala et al. 2012). In addition, while the genus period from spring to autumn. They spend the rest of Phymaturus is widely distributed in the cold and harsh the year in dormancy in rock crevices like other environments on both sides of the Andean Highlands Liolaemids lizards from Patagonia Argentina, such as (Argentina and Chile) and in the arid Patagonian Liolaemus elongatus (Ibargüengoytía and Cussac Argentinean steppe (Cei 1986, 1993), individual 1998; 1999), Liolaemus pictus (Ibargüengoytía and species have a very restricted distribution and this Cussac 1996; 1999), Liolaemus bibronii, Liolaemus genus exhibits significant microendemisms (Abdala et boulengeri, and Liolaemus lineomaculatus (Medina al. 2012). In the last 30 years, the number of known and Ibargüengoytía 2010). Most species of Phymaturus species has increased from 10 to 41 Phymaturus studied complete a female reproductive (Núñez et al. 2010; Troncoso-Palacios and Lobo 2012; cycle in two years (P. vociferator: Habit and Ortiz Lobo et al. 2013; Scolaro et al. 2013; Abdala and 1996; P. tenebrosus: Ibargüengoytía 2004; P. Quinteros 2014), and there are another 31 populations antofagastensis: Boretto and Ibargüengoytía 2006; P.

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Herpetological Conservation and Biology punae: Boretto et al. 2007; P. cf. palluma: Cabezas- precloacal pores in males), and measured each Cartes et al. 2010). The only exception occurs in specimen (digital gauge ± 0.02 mm, CA-01, Lee Phymaturus zapalensis females, which can reproduce Tools, Guangzhou, Guangdong, China) to study the annually but occasionally skip a year of reproduction allometric changes during ontogeny and sexual and therefore have an annual-biennial cycle (Boretto dimorphism. From the total sample of 178 lizards, we and Ibargüengoytía 2009). The cold harsh classified by abdominal palpation according to the environments that Phymaturus lizards inhabit, together reproductive state (see further explanation below) a with the physiological inability of viviparous females subsample of 151 individuals captured during the field to perform vitellogenesis while gravid (Duvall et al. trips of December 2010 and February-March 2011. 1982; Callard et al. 1992; Custodia-Lora and Callard We used GPS data (3 m resolution) to release most 2002), restrict the time available to complete a female lizards at their capture sites immediately following reproductive cycle in one year. Males of Phymaturus measure and palpation. Two groups of lizards were species have developed different ways of adapting to not immediately released. The first group consisted of female cycles such as prenuptial, postnuptial, or 12 gravid females that were transported to the continuous spermatogenic cycles. In species with laboratory until parturition (see further explanation prenuptial cycles, males perform spermatogenesis below), after which they were released at their original from spring to mid-summer, in synchrony with the capture sites. The second group consisted of the other follicular development and ovulation of females (P. 27 lizards of the total sample (17 males and 10 vociferator, Habit and Ortiz 1996; P. punae, Boretto et females). We brought these lizards to the laboratory, al. 2007). Postnuptial cycles are characterized by the where they were euthanized by intraperitoneal temporal dissociation between spermatogenesis and administration of sodium thiopental anesthetics. They mating, in which spermatogenesis lasts almost a year were then fixed in Bouin's solution 24 h and preserved from summer, autumn, and winter before resuming the in 70% ethanol to be used to study the spermatogenic following spring, when mating and ovulation occur (P. stages in males and to corroborate the reproductive tenebrosus: Ibargüengoytía 2004; P. zapalensis: state determined by palpation. For this work, we Boretto and Ibargüengoytía 2009; Boretto et al. 2012). followed the ASIH/HL/SSAR Guidelines for Use of Males with continuous spermatogenic cycles exhibit Live Amphibians and Reptiles as well as the sperm availability along the entire activity season from regulations detailed in Argentinean National Law spring to autumn (P. antofagastensis: Boretto and #14346. We deposited the specimens in the collection Ibargüengoytía 2006; Boretto et al. 2010; P. cf. of the Centro Regional Universitario Bariloche of the palluma: Cabezas-Cartes et al. 2010). Universidad Nacional del Comahue. In this study, we examined the reproductive cycles Phymaturus spectabilis habitat is located in the of males and females, minimum adult size, clutch size, Geomorphologic Region of Endorheic Depressions, in allometric changes during ontogeny, and sexual the arid district of the Monte Austral (Godagnone and dimorphism of Phymaturus spectabilis. This species Bran 2009). The dominant landscape is characterized is a microendemic to an area of approximately 70 km2 by barren steppe and dissected rolling plains in a volcanic rocky plateau at 1,000 m elevation, close interspersed with rocky outcrops and vegetation of a to Ingeniero Jacobacci, in the south-western area of shrub-steppe grassy appearance (Cabrera 1971; the Río Negro Province, Patagonia Argentina (Lobo Godagnone and Bran 2009). There is low herbaceous and Quinteros 2005; Pincheira-Donoso et al. 2008). In coverage, composed of sparse cushion bushes (Stipa addition, we show the accuracy of the non-invasive papposa, S. speciosa, S. humilis, Poa ligularis, and technique of abdominal palpation to determine the Bromus catharticus), and subshrubs (Senecio reproductive stage of adult females in this species with filaginoides, Nassauvia glomerulosa, and Grindelia the objective of promoting the use of non-invasive chiloensis) with bare soil percentages over 50% techniques in the study of lizards in Argentina that are (Cabrera 1971). We obtained the total daily vulnerable or at risk of extinction. precipitation (mm), and the maximum, minimum, and mean daily temperature from 1999 to 2011 from the METHODS National Meteorological Service of the Argentinean Air Force station, located in Maquinchao 70 km from Specimens used and capture site.—We captured P. Ingeniero Jacobacci. We show the biweekly means of spectabilis lizards from their endemic area, 25 km the total daily precipitation and the biweekly means of south of Ingeniero Jacobacci, Río Negro, Argentina the maximum, minimum, and mean temperature from (41°S, 69°W at 983–1,064 m elevation). We carried the last 13 years (Fig. 1). out field work during March 2006, November 2006, October 2007, December 2010, and February-March Abdominal palpation.—We palpated each 2011. During these field trips, we captured by individual (n = 151), which involved gently rolling slipknot and georeferenced 178 lizards in granite rock sections of the lizard’s abdomen between thumb and outcrops from 900 to 1900. We weighed (body mass, fingers to feel the size and shape of structures within BM, 100 g spring scale ± 0.5 g; Pesola AG, Baar, the ventral section of the body following Sinervo and Switzerland), determined sex (by the presence of Licht (1991). For each female we registered the

171 Boretto et al.—Reproductive Biology of Phymaturus spectabilis in Argentina.

30 18

25 16

14 20 12 15 10 10 8 5 6 Temperature (° C) Precipitation (mm) Precipitation 0 4

-5 2

-10 0 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec

FIGURE 1. Climatic conditions from the nearest meteorological station to the study site (Maquinchao, 70 km Northeast). Biweekly means of total mean precipitation (dash-dot) and maximum (solid), minimum (dotted) and mean (long dash) temperatures (°C) from January 1999 to 2011 are presented.

presence/absence of prominent spherical intra- condition using morphological observations of the abdominal structures indicative of vitellogenic ovarian follicles and uteri. Considering that follicles follicles, or the presence of prominent elliptical are spherical and that their diameter has been shown to structures indicative of intrauterine embryos. be a reliable variable to study the follicular growth Considering the abdominal palpation and the during vitellogenesis, we defined three follicular minimum adult size (defined below), we classified the categories. We used the largest follicular size (Duarte females as juveniles (lack of prominent spherical or Rocha 1992), measured with a vernier caliper on a elliptical intra-abdominal structures, and snout-vent camera Lucida scheme (± 0.1 mm), and yolk length [SVL] less than the minimum adult size), adult characteristics to classify the follicles as: vitellogenic females (presence of prominent spherical previtellogenic (small size and absence of yolk), intra-abdominal structures), gravid females (presence vitellogenic (larger size than previtellogenic follicles, of prominent elliptical structures indicative of and presence of yolk), or atretic (anomalous intrauterine embryos), or non-reproductive adult distribution of yolk). Finally, based on the integration females (lack of spherical or elliptical intra-abdominal characteristics of the reproductive tract (uteri) and the structures, and SVL more than the minimum adult follicular categories (sensu Ibargüengoytía 2004; size). For each male, we registered the Boretto and Ibargüengoytía 2006, 2009; Boretto et al. presence/absence of prominent spherical structures in 2007), we classified the females as juvenile (uteri like ventral section of body (indicative of development of a thread, transparent, without folds, and with pre- testicles) and presence/absence of combat marks. vitellogenic and small follicles), adult vitellogenic Considering the abdominal palpation and the female (distended uteri with medium-size folds over minimum adult size (defined below) we classified the the entire surface, with enlarged vitellogenic follicles), males as juveniles (lack of prominent spherical non-reproductive adult (distended uteri with medium- structures in ventral section of body, without combat size folds with small vitellogenic follicles), or post- marks, and SVL minor than the minimum adult size) partum adult female (conspicuous and expanded uteri, or adults (presence of prominent spherical structures in with numerous and large folds over the entire surface, ventral section of body, and combat marks). indicative of recent parturition). We classified the development stage of embryos sensu Leyton et al. Female reproductive condition, minimum size at (1980) from euthanized gravid females (n = 2) as: sexual maturity, clutch size, and mean annual early (from cleavage to somitic embryos), middle reproductive output.—We dissected the euthanized (curvate trunk to limbs with five fused fingers, females (n = 10) and determined their reproductive outlines of ears and eyes, and abundant yolk), and late

172 Herpetological Conservation and Biology

(with gonadal differentiation, scales and pigmented (BW, gravid females not included), diameter of the skin). front leg (FLD) and diameter of the hind leg (HLD) at We estimated the minimum SVL at sexual maturity the insertion with the shoulders and pelvic girdles, based on the size of the smallest female with any of respectively, right and left distance between front and the following traits explained above: follicles with hind limbs (inter-limb length, IL), and hip width yolk, embryos in uteri, or distended and folded uteri. (HipW) measured as the body width at the insertion of To estimate the dates of parturition and the clutch size hind legs. in P. spectabilis, during January-February 2011, we transported 12 gravid females to the laboratory and Statistical analyses.—We used the statistical maintained them in the laboratory until parturition. software Sigma Stat 3.5® (Systat Software Inc., We provided females with food and water ad libitum, Chicago, Illinois, USA), SPSS 17.0® (Chicago, in an open-top, glass terrarium (117 × 40 × 50 cm) Illinois, USA), and Sigma Plot 10.0® (Systat Software with sand and rocks from the site of capture, UV lamp Inc., Chicago, Illinois, USA). All data met the (Sylvania-Reptistar®, Erlangen, Bavaria, Germany), assumptions for parametric analysis unless otherwise and infrared lamp (150W, General Electric®, stated. For the analysis of the differences in SVL Budapest, Central Hungary, Hungary) on one side to between adult males and females, we used a t-test. provide a temperature gradient. Air temperature in the When the data did not meet the assumptions of room ranged from 18.8–23.1° C. We estimated the normality and homogeneity of variance required for clutch size by counting the total number of embryos parametric tests, we used the Mann-Whitney test. present in the right and left uterus of each female Furthermore, to avoid size dependence in the analysis euthanized, and the number of offspring born in of sexual dimorphism, we used the residuals from a captivity from each female. We calculated the mean regression of the natural logarithms (ln) of each annual reproductive output as the product of clutch morphological variable against ln(SVL) in a size and clutch frequency (1: annual female Discriminant Analysis. We analyzed the allometric reproductive cycle; 0.5: biennial reproduction; sensu changes along ontogeny by doing the regression of the Cree 1994; 0.75: annual-biannual reproduction; sensu ln of each morphometric variable and ln(SVL) in Boretto and Ibargüengoytía 2009). juveniles, adult males and adult females. For the analysis of the relationship between SVL and Male reproductive condition and sexual follicular or testicular size, or between reproductive maturity.—We measured the antero-posterior qualitative features in males, we used Linear diameter of testis of euthanized males (n = 17) with a Regressions or Pearson and Spearman correlations. vernier caliper on a camera Lucida scheme (± 0.1 We used the Paired t-test to analyze the differences mm). To study the reproductive cycle of males, we between right and left testicular size, and the binomial removed the right testis and epididymis and test to analyze the differences in the proportion of dehydrated in an ethanol series, and embedded in reproductive versus non-reproductive females, and in paraffin. We stained the sections (7 μm) with the proportion of reproductive-receptive males and Hematoxylin and Eosin (Martoja and Martoja Pierson females. We tested the assumptions of normality and 1970). We defined the stages of spermatogenesis homogeneity of variance with the one-sample sensu Mayhew and Wright (1970) as: (1) only Kolmogorov-Smirnov test and with the Levene test, spermatogonia; (2) primary and/or secondary respectively (Sokal and Rohlf 1969). Means are given spermatocytes; (3) spermatids; (4) spermatozoa in ± SE and for tests, α = 0.05. tubular lumen and in the epididymis; or (5) regression, with scarce spermatozoa in tubular lumen and sperm RESULTS in epididymis. We determined the minimum SVL at sexual maturity in males based on the size of the Minimum adult sizes, sexual dimorphism and smallest individual showing spermatogenesis or allometric changes along ontogeny.—The abdominal spermatozoa in epididymis. palpation allowed us to determine the minimum adult size of female P. spectabilis as 83.6 mm SVL, which Allometric changes during ontogeny and sexual presented prominent elliptical structures indicative of dimorphism.—For analyses of allometry, we included intrauterine embryos. The smallest euthanized female newborns (born in the laboratory, n = 20) and that showed signs of an adult reproductive condition, juveniles (n = 53). We restricted the morphological namely a distended uterus with medium-size folds descriptions for the assessment of sexual dimorphism over the entire surface, had a SVL of 83.1 mm. The only to adult males (n = 57) and females (n = 63) minimum adult size of a male showing reproductive based on the minimum adult sizes estimates from activity recognized by palpation was 77.4 mm SVL euthanized individuals. We used a digital gauge (± 0.2 and in a euthanized male that showed spermatozoa mm, Lee Tools, Guangzhou, Guangdong, China) to testicular stage was 79.6 mm SVL. In consequence, measure the following variables: SVL, head length we considered for the reproductive cycles, and for the (HL), head width (HW), neck width (NW), width of allometric and dimorphic studies that the minimum the tail base at the vent (WTV), maximum body width

173 Boretto et al.—Reproductive Biology of Phymaturus spectabilis in Argentina.

Adult males

Adult females

Juveniles

Newborns

40 45 50 55 60 65 70 75 80 85 90 95 100 105 Snout-vent length

FIGURE 2. Snout-vent length of newborns (squares), juveniles (diamonds), adult females (triangles), and adult males (circles) of Phymaturus spectabilis. The arrows indicate the minimum adult sizes obtained by abdominal palpation classifications for males (black arrow) and females (gray arrow).

adult SVL in females was 83.1 mm, and in males was captivity until parturition in midsummer (third and 79.6 mm (Fig. 2). fourth week of February) to two offspring each. In Comparison between males and females showed addition, in the field we captured newborn lizards also that females exhibited significantly larger SVL (nfemales in midsummer (47.5–49.5 mm SVL), and juveniles = 87, nmales = 85; U = 5,463.0, P < 0.001; meanfemales = with small SVL were captured in spring (rangeSVL = 89.8 ± 0.5 mm; meanmales = 86.0 ± 0.4 mm) and inter- 50.2–53.0 mm, n = 3), confirming that in the field, limb length than males, whereas males exhibited larger parturition occurs during the same period of time head width than females (Discriminant Analysis, λ = (midsummer) that were observed in the laboratory. 0.759, χ2 = 12.679, df = 2, P < 0.002). Confidence Females of P. spectabilis exhibited a fixed clutch size intervals of the allometric regressions of morphometric of two offspring per female. variables with SVL showed that HW, HL, NW, WTV, In early autumn, we captured two non-reproductive FLD, and HLD have a significantly higher growth rate females that exhibited small vitellogenic follicles in adult males than in females after sexual maturity, measuring 3.0 and 3.6 mm respectively, and two while females showed a significantly higher growth vitellogenic females with their largest vitellogenic rate only in IL (P < 0.050; Fig. 3). follicles measuring 6.0 and 11.9 mm, respectively (Fig. 4), and distended uteri with medium-size folds Female reproductive cycle, clutch size, and mean indicative of the medium vitellogenic stage. annual reproductive output.—In early spring, we Considering both euthanized and palpation captured one adult female at the early vitellogenic methodologies, Phymaturus spectabilis did not exhibit stage with its largest follicle measuring 3.6 mm, and significant differences in the general proportion of two post-partum females, with their largest follicle reproductive (follicles larger than 4 mm or gravid; n = size of 4.6 mm and 2.9 mm respectively (Fig. 4A). In 36, 59%) and non-reproductive females (follicles mid spring, we captured one non-reproductive female smaller than 4 mm or non-reproductive by abdominal with its largest follicle size of 2.2 mm, and two gravid palpation; n = 25, 41%; Binomial Test, P = 0.200, n = females with embryos in early stages of development 61). The SVL of reproductive and non-reproductive (Fig. 4A). From late spring to midsummer, we females did not differ significantly (meanSVL reproductive captured 49 juvenile females and 48 adult females = 91.1 mm; meanSVL non-reproductive = 90.3 mm; t = - classified by ventral abdominal palpation, and 58% of 0.684, df = 59 P = 0.496, n = 61). the adult females (n = 28) were reproductive (advanced vitellogenesis or gravid), whereas the other Male reproductive cycle.—Right versus left 42% (n = 20) were non-reproductive (Fig. 4A). The testicular size of adult males did not differ gravid females captured with external signs of significantly (t = 1.255, df = 15, P = 0.229, meanright = advanced gravidity (n = 12), and maintained in 7.6 ± 0.6 mm, meanleft = 7.4 ± 0.5 mm, n = 16).

174 Herpetological Conservation and Biology

4.0 2.9 3.0

2.9 3.8 2.8

A B 2.8 C 3.6 2.7 2.7 3.4 2.6 2.6 2.5 3.2 2.5 ln Head Width Head ln ln Head Length ln Head ln Body Width Body ln 2.4 3.0 2.4

2.3 2.8 2.3

2.2 2.2 2.6 3.6 3.8 4.0 4.2 4.4 4.6 3.6 3.8 4.0 4.2 4.4 4.6 3.6 3.8 4.0 4.2 4.4 4.6 ln SVL ln SVL ln SVL

2.8 3.4 2.2

2.6 D 3.2 E 2.0 F 2.4 3.0 1.8

2.2

2.8 1.6

2.0 Width ln Neck ln Front Leg Diameter Leg Front ln 2.6 1.4 1.8 ln Width of the Tail at the Vent the at Tail the of Width ln

1.6 2.4 1.2 3.6 3.8 4.0 4.2 4.4 4.6 3.6 3.8 4.0 4.2 4.4 4.6 3.6 3.8 4.0 4.2 4.4 4.6 ln SVL ln SVL ln SVL

2.4 3.2 4.0

2.2 G 3.0 H 3.8 I

2.0 2.8 3.6

1.8 2.6 3.4 ln Hip Width ln Interlimb Length Interlimb ln ln Hind Leg Diameter 1.6 2.4 3.2

1.4 2.2 3.0 3.6 3.8 4.0 4.2 4.4 4.6 3.6 3.8 4.0 4.2 4.4 4.6 3.6 3.8 4.0 4.2 4.4 4.6 ln SVL ln SVL ln SVL

FIGURE 3. Allometric relationships of the natural logarithms (ln) of all morphometric variables with SVL, for male and female Phymaturus spectabilis. Regression and confidence intervals are indicated (black: males; gray: females). Vertical lines indicate SVL at sexual maturity determined by gonadal index and histology of gonads (black: males; gray: females), the arrows indicate when a morphometric variable differs between males and females. A: ln maximum body width versus ln SVL; B: ln head width versus ln SVL; C: ln head length versus ln SVL; D: ln width of the tail base at the vent versus ln SVL; E: ln neck width versus ln SVL; F: ln front leg diameter versus ln SVL; G: ln hind leg diameter versus ln SVL; H: ln hip width versus ln SVL; I: ln interlimb length versus ln SVL.

Consequently, we arbitrarily chose the right testes for DISCUSSION the histological analyses. In the P. spectabilis adult males, there were no significant relationships between Females of P. spectabilis can display an annual testicular size and SVL (Log10 transformed; F1,16 = cycle in which vitellogenesis begins in autumn and 1.243, P = 0.282), but there was a significant finishes in mid spring and gravidity occurs from late relationship between testicular size and spermatogenic spring to midsummer. Nevertheless, the fact that the stage (r = 0.882, P < 0.001, n = 17). proportion of non-reproductive females did not At the beginning of spring males exhibited significantly deviate from 50% during the activity spermatozoa in testis and epididymis (stage 4, n = 3), season indicates that some females skip of a year of and in mid spring they had testis in regression and reproduction, and consequently, have a biennial abundant spermatozoa in the epididymis (stage 5, n = reproductive cycle. The presence of hypertrophied 6; Fig. 4B). In midsummer males began a new uteri with medium-size folds over the entire surface spermatogenic cycle, showing spermatocytes (stage 2, characteristic of estradiol activity and sexually mature n = 2) or spermatids in testis (stage 3, n = 1), some of females (Van Wyk 1991) allowed us to confirm that the individuals had traces of sperm in the epididymis, these individuals were adult females of P. spectabilis. from the previous cycle. In early autumn some males In addition, we can also discard the possibility that exhibited spermatids (stage 3, n = 4) and another there is a displacement of smaller females by larger exhibited spermatozoa in tubular lumen (stage 4, n = individuals as the non-reproductive adult females are 1), none of them had sperm in epididymis (Fig. 4B). not significantly smaller than those adult females, which are in the vitellogenesis or gestation stage. Operative sex ratio.—There were significant Based on these different lines of evidence, we defined differences in the proportion of reproductive males- the female reproductive cycle of P. spectabilis as receptive females (Proportion reproductive males – annual-biennial. Skipping a year has also been receptive females = 65–35; Binomial Test, n = 95, P < described for other species from diverse taxa linages 0.004). living in harsh environments, such as the lizard Cordylus giganteus (South Africa; Van Wyk 1991), the snakes Arizona elegans and Crotalus viridis (New

175 Boretto et al.—Reproductive Biology of Phymaturus spectabilis in Argentina.

our hypothesis that mating and ovulation in P.

Winter Spring Summer Autumn spectabilis occur during the first half of this season. Partum in captivity The same reproductive cycles have been observed in Gravidity 24 other Phymaturus that inhabit the Patagonian steppe of 22 20 Argentina, as P. tenebrosus (Ibargüengoytía 2004) and Vitellogenesis 18 and Ovulation P. zapalensis (Boretto and Ibargüengoytía 2009) 16 pointing out that there is a reproductive pattern of 14 postnuptial male cycles and annual to biennial female 12 cycles among the P. patagonicus group, which differs 10 8 from the P. palluma group (Boretto and 6 Ibargüengoytía 2009). Females from the P. palluma 4 Rest group are strictly biennial and males have exhibited 2

Largefollicular and size embryonic length(mm) different reproductive cycles from prenuptial to 0 September October November December January February March April continuous and asynchronous (Habit and Ortiz 1996; 58 % reproductive females 42 % non-reproductive females Boretto and Ibargüengoytía 2006; Boretto et al. 2007; Mating Period Cabezas-Cartes et al. 2010). In addition, P. spectabilis showed sexual 5 (6) dimorphism characterized by a larger SVL in females 4 (3) than males and a larger head width in males than females, as in the other species studied from the P.

3 (4) patagonicus group (Ibargüengoytía 2004; Boretto and

Ibargüengoytía 2009), whereas a larger SVL in males Spermatogenic Stages 2 (2) than females has been observed in all species of the P. palluma group (Boretto and Ibargüengoytía 2009; 1 Cabezas-Cartes et al. 2010). The larger heads in males

of P. spectabilis may have arisen through sexual September October NovemberDecember January February March April selection, in order to enhance success in male-male fights (Ibargüengoytía and Cussac 1999; Boretto and FIGURE 4. Reproductive cycle of males and females of Ibargüengoytía 2006), as a consequence of the male Phymaturus spectabilis. A: Large follicular sizes (mm; circles) of dissected adult females and length of embryos (mm; triangles) biased operational sex ratio, due to the low proportion from dissected gravid females versus date. The inferior bars of reproductive females in the population. The larger indicate the period of time when females were captured and inter-limb length observed in P. spectabilis females palpated and the percentage of reproductive and non-reproductive has been previously observed in all Phymaturus females determined by abdominal palpation. The dotted lines indicate the period of time when females undergoing each phase in studied, supporting that this dimorphic trait (larger in the reproductive cycle according to the information of dissected females than in males) is a characteristic of the genus females (triangles and circles data points) and palpated females (Boretto and Ibargüengoytía 2009; Cabezas-Cartes et (bars). B: Spermatogenic stages versus date of juveniles al. 2010; present study). In several species, a larger (spermatogenic stage 1) and adult males (spermatogenic stages 2 to 5) with abundant (square), moderate (triangle), or without inter-limb length in females can be attributed to spermatozoa in epididymis (circle). Brackets indicate the number fecundity selection for increased space to hold the of observations. developing eggs or embryos (Andersson 1994, Olsson et al. 2002), increasing not only the number of

Mexico; Aldridge 1979), the water skink Eulamprus offspring, but also offspring size (e.g., Shine 1992; tympanum (Schwarzkopf 1993) and the scincid Qualls and Shine 1995). Future studies will confirm Niveoscincus microlepidotus (Australia; Olsson and whether in Phymaturus there is a strong selection Shine 1998, 1999), among others. Skipping regarding embryo size or energetic reserves in reproduction has been also observed in Patagonia in newborns related to a larger inter-limb length, SVL or the lizards Liolaemus pictus (Ibargüengoytía and body width, a common pattern when the brood size is Cussac 1996), and P. zapalensis (Boretto and small (Olsson et al. 2002). In addition to the small Ibargüengoytía 2009), and in the nocturnal gecko clutch size (fixed at two newborns) and the prolonged Homonota darwini (Ibargüengoytía and Casalins annual-biennial female reproductive cycles, P. 2007). spectabilis exhibited a low mean annual reproductive Males of P. spectabilis, however, exhibited an output of 1.5 (2 × 0.75), and output for the other annual reproductive cycle synchronous with that of Phymaturus (0.75: P. vociferator, P. punae, P. cf. reproductive females. Males initiate the spermatogenic palluma; Habit and Ortiz 1996; Boretto et al. 2007; cycle in midsummer, and spermatogenic stages 3 Cabezas-Cartes et al. 2010; 1:0 P. tenebrosus, P. (spermatids) and 4 (spermatozoa) are present from antofagastensis; Ibargüengoytía 2004; Boretto and early autumn to the next spring. The presence of Ibargüengoytía 2006; 1.12: P. zapalensis; Boretto and males with spermatozoa in epididymis at the Ibargüengoytía 2009) are among the lowest for reptiles beginning of spring and the presence of females at in the world (Cree 1994; Ibargüengoytía and Casalins early gravidity at the end of spring strongly support 2007).

176 Herpetological Conservation and Biology

In spite of the differences in the timing of female example, the type locality of Phymaturus manuelae reproductive cycles among Phymaturus phylogenetic population is already locally extinct (pers. obs.). groups, in all the species studied, parturitions occur Herein, we established the efficacy of non-invasive during mid to late summer, revealing once again, the methodologies for the study of reproductive biology in constraints that harsh environmental conditions have P. spectabilis, particularly important due to the on the timing of reproduction. The cold environments vulnerable conservation status of the species and the of the Andes highlands and the Patagonian Argentina genus (Abdala et al. 2012). The knowledge of the where Phymaturus inhabit, together with hormonal patterns of reproduction, and specially the rates of and physiological restrictions inherent to viviparity reproduction, are relevant for conservation. The (Callard et al. 1992; Custodia-Lora and Callard 2002), possibility of studying the reproductive biology of prevent females from completing vitellogenesis, species using non-invasive techniques is a critical tool. gravidity, and storing fat within one activity season Abdominal palpation used in P. spectabilis is an (Boretto et al. 2007; Boretto and Ibargüengoytía 2009; effective technique to discriminate juveniles from Cabezas-Cartes et al. 2010). Under this scenario, adults, reproductive stages of adult females and size at when the length and thermal quality of the activity sexual maturity, and confirmed the plasticity of an season are variable, flexibility in frequency of annual-biennial female cycle without the need to reproduction seems to be an efficient mechanism to euthanize a larger number of lizards. Indeed, the cope with unpredictable climates (Boretto and minimum adult size determined by palpation, was Ibargüengoytía 2006, 2009). When the activity supported by the allometric changes observed between seasons are relatively cool and/or short and postpartum juvenile and adults. In summary, it is necessary to feeding opportunities are limited, females devote the develop non-invasive programs and bio-ecological following season to growing and storing reserves (e.g., studies of this poorly known genus to protect the capital breeding) for future reproductive bouts, but herpetofauna of Patagonia. under benign conditions reproduction can be resumed in a year (Saint Girons 1985; Schwarzkopf 1993; Cree Acknowledgments.—We wish to express our 1994; Ibargüengoytía and Cussac 1996; Boretto and gratitude to Joel A. Gutiérrez and Erika L. Kubisch for Ibargüengoytía 2009). Parental “allocation decisions” their assistance during fieldwork, and to the National that increase an offspring’s probability of survival Meteorological Service and Argentinean Air Force for through the earliest stages of their life cycle are under providing climate data. This study was conducted strong selection pressure (Sinervo et al. 1992), and with research grants from CONICET (PIP 00033) and may counteract the low frequency of reproduction FONCYT (PICT 1125) with capture permit from the resulting from multiannual reproductive cycles. The Dirección de Fauna de Río Negro Province. Barry abundant fat reserves observed in the offspring of P. Sinervo was supported by NSF grant IOS-1022031. spectabilis, as well as the high parental investment per offspring denoted by the large fat bodies and intra- LITERATURE CITED abdominal yolk reserves found in the offspring of P. punae (Boretto et al. 2007), P. antofagastensis, P. cf. Abdala, C.S., and S.A. Quinteros. 2014. Los últimos palluma (Cabezas-Cartes et al. 2010) and P. 30 años de estudios de la familia de lagartijas más zapalensis (Boretto and Ibargüengoytía 2009), diversa de Argentina. Actualización taxonómica y reinforces this prediction, because these reserves could sistemática de Liolaemidae. Cuadernos de enhance offspring survival, even when the offspring´s Herpetología 28:In Press. investment might be detrimental for future Abdala, C.S., J.L. Acosta, J.C. Acosta, B. Alvarez, F. reproduction of females (Boretto et al. 2007; Boretto Arias, L. Avila, G. Blanco, M. Bonino, J.M. Boretto, and Ibargüengoytía 2009; Cabezas-Cartes et al. 2010). G. Brancatelli, et al. 2012. Categorización del estado The P. patagonicus group is especially vulnerable de conservación de los lagartos de la República because the isolated populations are restricted to rock Argentina. Cuadernos de Herpetología 26:215–248. promontories of only severely fragmented habitats in a Aldridge, R.D. 1979. Female reproductive cycles of limited geographic range (approximately 700 km2; snakes Arizona elegans and Crotalus viridis. Piantoni et al. 2006; Abdala et al. 2012). In addition, Herpetologica 35:256−261. the habitat is going through a desertification process, a Andersson, M. 1994. Sexual Selection. Princeton significant anthropogenic impact caused by the over- University Press. Princeton, New Jersey, USA. exploitation of the land, especially by sheep ranching Boretto, J.M., and N.R. Ibargüengoytía. 2006. and shrub fires often occur in summer (Piantoni et al. Asynchronous spermatogenesis and biennial female 2006), aggravated by global climate change (Sinervo cycle of the viviparous lizard Phymaturus et al. 2010). As a consequence, Phymaturus antofagastensis (Liolaemidae): Reproductive populations are showing retraction and some may have responses to high altitudes and temperate climate of become extinct, probably because of their viviparous Catamarca, Argentina. Amphibia-Reptilia 27:25–36. condition and their preference for low body Boretto, J.M., and N.R. Ibargüengoytía. 2009. temperatures (Ibargüengoytía and Casalins 2007). For Phymaturus of Patagonia, Argentina: Reproductive biology of Phymaturus zapalensis (Liolaemidae)

177 Boretto et al.—Reproductive Biology of Phymaturus spectabilis in Argentina.

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179 Boretto et al.—Reproductive Biology of Phymaturus spectabilis in Argentina.

JORGELINA M. BORETTO is an Assistant Investigator of CONICET, and conducts research in the Laboratory of Eco-physiology and Life History of Reptiles of the Department of Zoology at INIBIOMA (CONICET-Universidad Nacional del Comahue), San Carlos de Bariloche city, Río Negro Province, Argentina. She has a Ph.D. from Centro Regional Universitario Bariloche of Universidad Nacional del Comahue. Her research interests include the study of reproductive biology, environmental endocrinology, and conservation of lizards of the genus Phymaturus and others liolaemids from cold climates of Patagonia Argentina. (Photographed by Carla Piantoni)

FACUNDO CABEZAS-CARTES is a Doctoral Scholarship Fellow of Consejo Nacional de Investigaciones Cientificas y Tecnicas (CONICET) at Instituto de Investigaciones en Biodiversidad y Medioambiente (INIBIOMA) in Bariloche, Argentina. He graduated with a degree in Biology from the Universidad Nacional de Cordoba and is currently a Ph.D. student at the Universidad Nacional del Comahue. His research interests include the ecophysiological adaptations to harsh climates, biogeography, and conservation of Phymaturus lizards. (Photographed by Jorgelina Boretto)

FABIÁN TAPPARI is an Assistant Teacher of Pollution of Soils and Waters at the National University of the Patagonia San Juan Bosco in Trelew, Chubut, Argentina. He has a degree in Protection and Environmental Reparation from the National University of the Patagonia San Juan Bosco and he is working on Ph.D. studying the natural history of two syntopic populations of the lizard genus Phymaturus. In addition, he is working in the technical area of Rawson's Zoo (Chubut), designing and preparing didactic exhibitions about reptiles and insects. (Photographed by Mary Villegas)

FAUSTO R. MÉNDEZ -DE LA CRUZ is a Professor at the Universidad Nacional Autónoma de México in México City. He got his Ph.D. in 1989 at the Universidad Nacional Autónoma de México and did postdoctoral work at the University of Florida, USA, and a sabbatical at Virgina Polytechnic Institute and State University, Virginia, USA. His research is focused on the evolution of reproductive systems in reptiles, mainly in the evolution of viviparity and parthenogenesis, and more recently in the vulnerability of amphibians and reptiles to climate change. (Photographed by Juan Carlos Cruz)

BARRY SINERVO is a Professor, Level VI, of Ecology and Evolutionary Biology, University of California, Santa Cruz. His research interests include behavioral ecology, effects of natural and sexual selection on reproduction, reptilian communities, speciation, and the effects of climate change on reptile populations. (Photographed by Erika Kubisch)

JOSE ALEJANDRO SCOLARO is an Emeritus Professor at the Universidad Nacional de la Patagonia San Juan Bosco in Trelew City. He got his Ph.M. in 1983 at the University of Bradford, United Kingdom, and his Ph.D. in 1984 at the Universidad de Córdoba, Spain. He has been researching and teaching for 46 y in several universities. His research is focused on lizards, especially on taxonomy, adaptive ecology, population dynamics, conservation, and more recently on vulnerability to climate change. (Photographed by Rafael Lara Résendiz)

NORA R. IBARGÜENGOYTÍA is a CONICET Researcher and a Chair of the Laboratory of Eco- physiology and Life History of Reptiles of the Department of Zoology at INIBIOMA (CONICET- Universidad Nacional del Comahue), S.C. de Bariloche, Argentina. Her major interest is in the eco-physiological evolution of squamate reptiles. Her research has focused largely on reproductive biology, thermal physiology, and the effects of climate change on reptile populations. Her current projects include studies on physiological responses to cold climate, evolution of viviparity, and effects of climate change on the genus Phymaturus, Liolaemus, and Tropidurus. (Photographed by Carla Piantoni)

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